Convergent community structure of algal–bacterial consortia and its effects on advanced wastewater treatment and biomass production

Microalgal-bacterial consortium is an effective way to meet increasingly stringent standards in wastewater treatment. However, the mechanism of wastewater removal effect has not been properly explained in community structure by phycosphere. And little is known about that the concept of macroecology was introduced into phycosphere to explain the phenomenon. In the study, the algal–bacterial consortia with different ratios of algae and sludge were cultured in same aerobic wastewater within 48 h in photobioreactors (PSBRs). Community structure at start and end was texted by metagenomic analysis. Bray–Curtis similarities analysis based on microbial community showed that there was obvious convergent succession in all consortia, which is well known as “convergence” in macroecology. The result showed that Bray–Curtis similarities at End (overall above 0.88) were higher than these at Start (almost less than 0.66). In terms of community structure, the consortium with 5:1 ratio at Start are the more similar with the consortia at End by which the maximum removal of total dissolved nitrogen (TDN, 73.69%), total dissolved phosphorus (TDP, 94.40%) and NH3-N (93.26%) in wastewater treatment process and biomass production (98.2%) higher than other consortia, according with climax community in macroecology with the highest resource utilization than other communities. Therefore, the macroecology can be introduced into phycosphere to explain the consortium for advanced wastewater treatment and optimization community structure. And the study revealed a novel insight into treatment effect and community structure of algal–bacterial consortia for advanced wastewater treatment, a new idea for to shortening the culture time of consortium and optimize predicting their ecological community structure and predicting ecological community.

Most suspended solids and organic pollutants can be removed by primary and secondary treatment of wastewater. Then the removal effect of nitrogen and phosphorus needs additional tertiary treatment to realize nitrogen and phosphorus removal, which increases the cost of wastewater treatment. Wastewater treatment needs additional energy and steps to realize nitrogen and phosphorus removal, which increases the cost of wastewater treatment 1 . The cost of aeration in the activated sludge process accounts for 45-75% of the cost of wastewater treatment 2 . In this process, the emissions of carbon dioxide and nitrogen oxide also caused environmental pollution [3][4][5] . In addition, excess sludge is also a "pollution transfer" 2 .
Microalgae has overcome the limitations of traditional wastewater treatment methods 6,7 . In recent years, the research on the absorption and transformation of nitrogen and phosphorus by the symbiotic system of algae and fine bacteria has aroused widespread concern 8 . The algal-bacterial consortium system has been favored by scholars due to its unique characteristics, such as reduced power consumption and biomass refinability 8 . Its principle is to use the relationship between algal bacteria and community structure to promote the effect of removing organic pollutants by heterotrophic bacteria and N, P by algae from wastewater 4,9 . Aeration provides excess oxygen to COD-degrading bacteria. Obviously, oxygen provided by algae photosynthesis is not useful for bacteria to remove COD. Therefore, bacteria have a dominant position in the community in the group with high COD removal efficiency. For example, the high algae/sludge ratios constitute algal-dominated algal-bacterial consortia which remove the nitrogen and phosphorus by algae absorption 10  www.nature.com/scientificreports/ The working volume of the reactor is 3 L, which is made of transparent organic glass. The reactor has a height of 300 mm, the inner diameter of 120 mm, and the wall thickness of 5 mm. The bottom of the reactor is provided with an air inlet which is connected with the aeration head. The top of the reactor is respectively provided with an air outlet and a spare feed inlet. The photobioreactor is shown in Fig. 1.
The experiments were conducted with mature algal-bacterial consortia in 5 uniform photobioreactors. The initial biomass concentration was 500 mg L −1 ; The temperature was 24 degrees; The 95:5 airflow-CO 2 (v/v) gas mixture into the bottom of the PSBRs with a speed of 0.6 L min −1 for each column to agitate the algal broth as well as supply carbon from air. The rubber-pipes were connected with glass connector to convey to the aeration head. The purity of CO 2 was 99%. The average illuminance was 4000 Lux. The period of batch operation was set at 48 h.
Analytical methods. In each cycle, samples were collected at intervals of 8 h, and then centrifuged at 4000 r/min for 10 min. The supernatants of samples filtered through 0.45 mm filter for further analysis, and were used for detecting Chemical oxygen demand (COD) determined by Dichromate method (HJ 828-2017), Total dissolved phosphorus (TDP) determined by Ammonium molybdate spectrophotometric method (GB 11893-89), Total dissolved nitrogen (TDN) determined by Alkaline potassium persulfate digestion UV spectrophotometric method (HJ 636-2012) and Ammonia nitrogen (NH 3 -N) determined by Nessler's reagent spectrophotometry (HJ 535-2009). The standard of methods were determined according to the national standards (Chinese state standard monitoring method) 27 .
The pH was determined to use a PHS-3B pH meter. The biomass concentration was estimated by dry weight (DW) measurement 26 . Each sample was set in triplicate to eliminate unexpected errors.
Microbial structure analysis. Microbial samples were collected at the start-up stage (Start, the 0 h) and the end of operation stage (End, the 48th h), and then stored at − 80 °C until the genomic DNA was extracted. According to manufacturer's agreement, microbial DNA was extracted by using the Tiangen DP329 DNA Kit (Tiangen Biotech, Beijing, China). The concentrations and purity of DNA samples were detected by 1% agarose gel electrophoresis. The extracted DNA were amplified by Polymerase Chain Reaction (PCR) (98 °C predegeneration for 1 min, followed by 30 cycles at 98 °C for 10 s, 50℃ for 30 s, 72 °C for 30 s and a final extension 72 °C for 5 min). For metagenomics analysis, Illumina Truseq DNA PCR-Free Library Preparation Kit was used to construct the Library 28 . After Qubit quantification and Library test, Novaseq 6000 was used for online sequencing.
Similarity analysis of community structure. The Bray-Curtis similarity was shown via Eq. (1).
where S A,i , S B,i , are counts of i species in community A and community B, respectively. The range from 0 to 1. The closer the value is to 1, the more similar the two communities become. On the contrary, the closer to 0, the greater the difference between the two communities. Analysis of similarities (ANOSIM), based on Bray-Curtis coefficient, may visualize the coefficients and show its significance. The R value was shown via Eq. (2).
where r b , r w , are counts of mean rank of between and within group dissimilarities, respectively. The R in the range of − 1 to 1. When R > 0 , it is significant in group. When R < 0 , the group is meaningless.

Results
Wastewater remediation. Figure 2 showed the nutrient removal of R1 to R5 in PSBR.  Table 1). The PSBR with R2 exhibited the highest and fastest NH 3 -N removal, compared with other consortia. The TDP removal in these complexes was also investigated. The fastest and highest TDP removal was found in these consortia   Table 1). The fastest and highest COD removal efficiency was found in these complexes inoculated with R5. It was obvious that the COD removal efficiencies with R1 and R5, whose difference in the amount of sludge between them. It's worth noting that the nutrient removal not only is related to the algae concentration, but also high concentration activated sludge affect the removal efficiency at initial stage (N and P removal efficiency R5 > R3 > R4). Within 48 h, the pH value of all the consortia increased. The maximum pH was R3 and minimum was R5. The range of pH was 8.5 to 10.5. After 32 h, the pH value was relatively stable.
Biomass accumulation. Figure 3 shows the increase in the biomass concentration. The biomass concentration of algal-bacterial consortia increased significantly after the absorption of nutrients. The biomassproductivity of R1 to R5 was 72.2%, 98.2%, 89.80%, 69.00% and 62.40% respectively (Table 1). Obviously, the highest growth efficiency of biomass was R2. The biomassproductivity of the complex with high algae concentration were higher than that of the complex with high activated sludge. However, the biomass productivity of R2 (98.2%) was higher than that of R1 (72.2%), and the algae concentration than R2 is higher. On the contrary, the biomassproductivity of the consortium of R4 (69.00%) was higher than R5 (62.40%) in high algae concentration.
The 24th h was an important node. After that, the efficiency of increasing biomass concentration has been improved significantly. The time point for nitrogen removal efficiency improvement was also 24 h. However, TDP removal effect was bigger and faster before the 18th h.
Convergence of community structure. Microbial community structure at Start. As shown in Fig. 4 and Table S1, the composition of native algae community is mainly Chlorella sp. and Scenedesmus sp. by the microscope of R1 to R5. At the same time, some cyanobacteria, such as, Anabaena sp. and Oscillatoria sp. have also been observed with all consortia.
Microbial community structure at End. At End, the community related to nutrient removal declined in bacteria community with all consortia. For example, the bacteria community related to denitrification process (Table S1), such as Pseudomonas, changed to weak community, down to 0.22%, 1.92%, 0.49%, 0.40% and 0.67%, respectively (Table S1). However, the ratio of algae community related to photosynthesis and bacteria community about heterotroph was 10.10:1, 6.58:1, 6.32:1, 6.06:1 and 5.05:1 respectively ( Table 2).
Convergence of community succession. All consortia with R1 to R5 at End was similarity to each other by Bray-Curtis similarity, which the succession of community from R1 to R5 has convergence. The Bray-Curtis simi-   (Table S1), got tiny changed with the process of wastewater treatment which was named SDJZ-CC1.   (1) to (19).

Discussion
Introduction of macroecology theory into phycosphere. The theories of phycosphere space-time evolution was only guided by the judgement of microbiologist Beijerinck that "environmental conditions determine microbes" 34 . In fact, phycosphere is a complete ecosystem 35 . Inorganic components including matter and energy, while producers, consumers and decomposers are composed of microbes 36 . Therefore, the macroecology should be introduced into revealing the mechanism of algae-bacterial consortium in phycosphere. At first, the community succession refers to the adjustment of the community structure and relationship between algae and bacteria under the influence of ecological factors such as nutrition, DO, pH, temperature and light 37 . The result of community succession is the Climax Community in which the most complex community structure and the most diverse community relationships exist under the specific nutrient conditions in wastewater 19 . The efficient use of nutrients is maximized in climax community in phycosphere. There is an optimum treatment effect to remove C, N and P for the algal-bacterial consortium in climax community. Therefore, the component with the best treatment effect in wastewater existed the climax community in secondary effluent. R2 gradually succeeded to the climax community in secondary effluent treatment process. Producers (autotrophs) are composed of microalgae or autotrophs 38 . Producers, such as algae, cyanobacteria or photosynthetic bacteria, can absorb the light energy and inorganic carbon in wastewater and convert it into organic matter to fix into the algal-bacterial consortia by photosynthesis 39 . Inorganic nitrogen is brought into organic nitrogen by nitrifying bacteria, such as Nitrospira. Producers convert inorganic carbon or inorganic nitrogen into organic forms in the phycosphere. Organizers participate in material circulation and signal transmission of the consortium. For example, the microalgae fix the inorganic carbon into organic carbon that is oxidized by bacteria to get energy. The CO 2 produced by bacterial oxidation is carbon source for photosynthesis of algae. The Decomposer can convert organic matter into inorganic matter. For example, Acinetobacter, the aerobic bacteria which can remove COD, oxidizes the organic carbon source into CO 2 to the growth of algae.
The community convergence is a product of community succession in the phycosphere. It is universally accepted that resources determine the direction of community convergence and there is only one climax community where resource and environmental conditions are established. Dominant populations dominate resources of the ecosystem. The number of competitors is limited and co-living may promote each other's growth. Community succession needs to be proven to be utilized by Community similarity analysis which needs statistical tools. Bray-Curtis similarity is often used by ecologists to quantify differences between samples based on abundance or counting data. In essence, it refers to a quantitative value, the size of which reflects the difference in community species composition between different samples within each group 20 . Bray-Curtis similarity clustered similar DNA sequences into a smaller number of taxa to improve the efficiency of analysis and analytical accuracy 24 .
Driving force of community succession. Nutrients (resource) drive the direction of succession of communities 39,40 . The level of nutrients determines the end point of the development of the ecological community 19 . And the ecological factors determine the development process of ecological community 39 . In this study, great utilization of nitrogen and phosphorus and aerobic environment caused by the photosynthesis and aeration led to autotrophic succession 26 . Therefore, convergence of succession occurs commonly in all consortia in the study (Table 4, Fig. 3). The efficiency of resource utilization by the community is consistent with the  40 . In other words, the climax community was the destination of the algal-bacterial consortium whose nitrogen and phosphorus removal were fastest and highest with all consortia. Therefore, the initial concentration of R2 would eventually develop into the climax community (Table 4). In aquatic ecosystem, the similarity tendency got reappearance 41 . For example, Cruz and Pompeu 41 revealed The importance of variables linking habitat diversity and basin connectivity to fish assemblage richness and structure by Bray-Curtis coefficients. And the similarity of fish assemblage community structure changes with time and space was also studied. In addition, there are similar studies in microecology and the distribution of the functional genes in suspended activated sludge system, such as antibiotic resistance genes 22,24 , was tested by Bray-Curtis coefficients. Convergence and climax community can deduce the optimal proportion of algae and bacteria in the initial community. As shown in Table 4, the Bray-Curtis similarity index of R2 at Start was closer to each component of End, especially the high proportion of sludge, such as R4 and R5. It was indicated that the community was succeeding in the direction closer to R2. However, R2 developed to the Climax Community faster when using environmental resources. Because R2 at Start had better ability of assimilation of nitrogen and phosphorus and coordinated symbiosis between biological communities. This result can be used to select appropriate algae and bacterial consortia to optimize wastewater removal efficiency and improve the biomass production capacity. The SDJZ-CC1 exists in an aerobic environment and its dominant species are autotrophs, such as microalgae community. This community structure leads to high nitrogen and phosphorus assimilation efficiency and biomass growth capacity.
Effects of community structure on wastewater treatment process. The autotrophs or heterotrophic microorganisms with the largest number in the climax community are called the dominant species 42 . The dominant species play an important role in the removal of nutrients 42 . In autotrophic microorganism dominant species, the removal of TDP was independent of the bacteria community since phosphorus accumulating bacteria can become dominant species in the alternate of aerobic and anaerobic process (Table 3) 25 . In addition, P removal was relevant to the assimilation by algae in aerobic autotrophic environment. Besides, in high pH values environment, which caused by photosynthesis, P can chemically be precipitated and be removed in wastewater. Therefore, there was little direct effect for bacteria community on TDP removal 43 . And the effect of removal TDP of consortium is relevant with a high proportion of algae 43 . However, excess algae can obscure the light-exposed area in the reactor, which impedes the ability of algae to absorb TDP 10 . The low proportion of bacteria inhibited the symbiosis between algae and bacteria 10 . It is generally believed that the symbiotic relationship between alga and bacteria promotes TDP absorption by algae 9 . The similarity is that nitrogen was removed by an algal-bacterial consortium. In this study, nitrogen removal is through assimilation of microalgae communities, because denitrification must belong to anaerobic bacteria in facultative anaerobic environment, rather than under aerobic condition (aeration or photosynthesis) 26 . In addition, the increasing of pH, due to the photosynthesis, affected the NH 3 -N removal. With pH value increasing, the free ammonia can be formed and volatilized. Therefore, nitrogen removal is related to the growth of microalgae 44 . The growth of microalgae is related to nitrogen absorption. Removal of NH 3 -N is preferred over TDN due to nitrification by ammonia-oxidizing bacteria, converting NH 3 -N to nitrate nitrogen which is good for absorption 45 . The growth of microalgae community increased the ability to absorb nutrition. The microalgae community was the dominant Species which gives priority to nutrition (Fig. 4). Therefore, competitive relations which occur between microcystis and alginolytic bacteria 46 may arise between microalgae communities and nitrifying bacteria, such as Pseudomonadales, Nitrospira (Fig. 4). The ratio of Chlorella and Scenedesmus increased due to the better absorptive capacity of Chlorella than Scenedesmus to produce bio-oil (Fig. 4). The similarity lies in the growth of Anabaena 47 and Oscillatoria 31 , belonging to cyanobacteria, decreased due to the competition among the microalgae community. In connection with previous literature, Liang et al. 48 achieved higher removal efficiencies of NH 3 -N (86.0%) and TDP (93.0%). In a similar wastewater environment (about NH 3 -N 20 and TDP 4 mg L −1 in the initial), the results obtained www.nature.com/scientificreports/ from the present study indicated the higher removal efficiencies of NH 3 -N ( > 85.0% and the study 93.26%) and TDP ( > 90.0% and the study 94.4%). As shown in Table 3, the dominant Species, in heterotrophic microorganism species, is Proteobacteria_unclassified and other bacteria belonging to Proteobacteria which has the ability to remove organic matter, nitrogen and phosphorus. However, the ability of Proteobacteria to remove nitrogen and phosphorus was not significant because the dominant Species of nitrogen and phosphorus was Chlorella. Therefore, the ability of Proteobacteria, such as α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria, to remove organic matter was significant 29 . The CO 2 and low molecular weight organic matter produced by Proteobacteria oxidation are provided to microalgae community. Therefore, COD is mainly oxidized by the bacterial community rather than microalgae community. And the growth of the community structure of the bacterial community about nitration, denitrification and release of phosphorus was decreased.
It is generally acknowledged that bacterial communities prefer acidic environments while microalgae communities thrive in alkaline environments. The increase of pH directly led to the change of community structure straightly (Fig. 3). Generally, the pH of the water environment where consortia of algae and bacteria live increases with the decrease of CO 2 concentration. The reason why the ratio 5:1 of the consortium was the highest is that symbiosis enhances the ability of the consortium to absorb CO 2 49 . Similarly, Su et al. 10 conducted a similar study on the highest nitrogen and phosphorus removal efficiencies observed with 5:1 for ratio of algae and sludge consortium, 91% and 93.5% respectively.
Effects on biomass production. The growth of biomass is related to carbon source and nutrition recovery 8 . Biomassproductivity and quality determine that the cost can be offset in the commercial application of wastewater treatment based on microalgae wastewater treatment 8 . And production of biomass is the most direct expression of consortium's resource utilization 50 . The biomass productivity of R2 was the largest among all consortia (Fig. 2). The removal of nutrients is also the most efficient (Fig. 3). Microalgae biomass was gradually accumulated, due to nitrogen supply shifted from N-rich to N-deficient condition was the absorption of algae with aeration oxygenation 26 . Therefore, the removal of nitrogen and the generation of algae biomass can be carried out simultaneously. However, the removal of TDP precedes the growth of biomass, because there may be a buffer period for the assimilation of TDP by algae 43 . In connection with previous literature, the results of this study show that the biomass production was related to the concentration of algae, with a high ratio of algae/ sludge at consortium 51 . The balance of victory was in favor of microalgae community, due to the dominance of their dominant community. In the microalgae community, Chlorella is considered to be a kind of quality algae to produce oils 25 . Novel insight in consortium for wastewater treatment and community structure. This study first explored that theory of ecology was introduced into phycosphere. Convergence of community succession in algal-bacterial consortia was demonstrated by Bray-Curtis similarity. The result of nutrients removal in consortia was consistent with the resource utilization in macroecology. In community structure, the algae, the role of productors, showed a high potential for biomass production by utilizing various inorganic carbon substrates on photosynthesis in aerobic autotrophic environment. Besides, bacteria, the role of consumers or decomposers, oxidized organic matter to promote the inorganic carbon substrates. Above all suggest that macroecology perspectives can explain community structure in phycosphere. Therefore, convergence of community succession in algal-bacterial consortia were explained based on macroecology. This is of great significance for exploring the shortening of community culture time and predicting ecological community.

Conclusions
In this study, the 'convergence' of community structure in the algal-bacterial consortia with different ratios of algae/sludge was analyzed confirmed by metagenomic sequencing and Bray-Curtis similarity, and 'convergence' which is well known as in macroecology was the succession results of community succession in macroecology the algal-bacterial consortia. It means that stable and similarity climax communities have eventually established in wastewater, no matter what the ratio of algae/sludge was in the initial. The consortium with 5:1 ratio of algae/sludge, which community structure was most similar to that at end, namely the climax community. The consortium, and achieved the highest nitrogen and phosphorus removal efficiency and biomass production. For this phenomenon ecology, it's the best ecological explanation that the highest resource utilization (nutrient contaminant uptake) climax community can be accessed. Therefore, the novel insight based on authenticated the theory well-established in macroecology introduced into the phycosphere can be employed to optimize their community structure to enhance shorten culture time of consortium for advanced wastewater treatment.